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The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells.

Feres KJ, Ischenko I, Hayman MJ - Oncogene (2008)

Bottom Line: In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors.However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells.Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY, USA.

ABSTRACT
The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

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Related in: MedlinePlus

RON exhibits MSP-independent tyrosine phosphorylation in 10A cells(a) GFP-positive cells were selected by FACS analysis to yield a 10A/Vector control cell line and RON-expressing cells. RON-expression levels were detected by Western blot with an anti-RON antibody. This antibody recognizes the three different processed forms of RON: the 170 kDa unprocessed form, the 150 kDa processed and phosphorylated form and the 145 kDa processed and unphosphorylated form. Total MAPK level was used as a loading control. (b) 10A/Vector and 10A/RON cells were immunoprecipitated with an anti-RON antibody and subjected to a Western blot analysis for phospho-tyrosine (4G10 antibody). The blot was reprobed for total RON expression. (c)The same experiment as in (b), blotting with antibodies against catalytically activated RON (pY1238/Y1239), and specific tyrosine phosphorylated docking sites in the C-terminus of RON (pY1353, pY1360). (d) Semi-quantitative RT-PCR detected MSP mRNA levels. COS1 cells were transfected with a vector containing an MSP cassette as a positive control. These experiments were repeated at least three times, and shown here are representative images.
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Figure 1: RON exhibits MSP-independent tyrosine phosphorylation in 10A cells(a) GFP-positive cells were selected by FACS analysis to yield a 10A/Vector control cell line and RON-expressing cells. RON-expression levels were detected by Western blot with an anti-RON antibody. This antibody recognizes the three different processed forms of RON: the 170 kDa unprocessed form, the 150 kDa processed and phosphorylated form and the 145 kDa processed and unphosphorylated form. Total MAPK level was used as a loading control. (b) 10A/Vector and 10A/RON cells were immunoprecipitated with an anti-RON antibody and subjected to a Western blot analysis for phospho-tyrosine (4G10 antibody). The blot was reprobed for total RON expression. (c)The same experiment as in (b), blotting with antibodies against catalytically activated RON (pY1238/Y1239), and specific tyrosine phosphorylated docking sites in the C-terminus of RON (pY1353, pY1360). (d) Semi-quantitative RT-PCR detected MSP mRNA levels. COS1 cells were transfected with a vector containing an MSP cassette as a positive control. These experiments were repeated at least three times, and shown here are representative images.

Mentions: Endogenous levels of RON were low in the relatively normal epithelial cell line, MCF-10A. Consequently, the cells did not respond to MSP in any biological or biochemical assays we have tested to date. Therefore, to examine the contributions of RON to the progression of breast cancer, we transduced MCF-10A cells with a retrovirus expressing wild-type human RON and IRES-promoted GFP. Pools of infected cells were selected for high, medium and low GFP expression levels by FACS, and, as expected, GFP levels were mimicked by expression levels of RON (Figure 1a). We designated these cells as 10A/RON and our vector-only negative control cell line was designated 10A/Vector. The levels of RON expression in 10A/RON cells were well within the range of expression found in human breast carcinomas, some of which had RON levels that were up to 72-fold higher than the level found in benign tissue (Maggiora et al., 1998). High, medium and low 10A/RON cell lines behaved similarly in all of our experiments therefore, we utilized the medium-RON expressing MCF-10A cell line throughout this study.


The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells.

Feres KJ, Ischenko I, Hayman MJ - Oncogene (2008)

RON exhibits MSP-independent tyrosine phosphorylation in 10A cells(a) GFP-positive cells were selected by FACS analysis to yield a 10A/Vector control cell line and RON-expressing cells. RON-expression levels were detected by Western blot with an anti-RON antibody. This antibody recognizes the three different processed forms of RON: the 170 kDa unprocessed form, the 150 kDa processed and phosphorylated form and the 145 kDa processed and unphosphorylated form. Total MAPK level was used as a loading control. (b) 10A/Vector and 10A/RON cells were immunoprecipitated with an anti-RON antibody and subjected to a Western blot analysis for phospho-tyrosine (4G10 antibody). The blot was reprobed for total RON expression. (c)The same experiment as in (b), blotting with antibodies against catalytically activated RON (pY1238/Y1239), and specific tyrosine phosphorylated docking sites in the C-terminus of RON (pY1353, pY1360). (d) Semi-quantitative RT-PCR detected MSP mRNA levels. COS1 cells were transfected with a vector containing an MSP cassette as a positive control. These experiments were repeated at least three times, and shown here are representative images.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628450&req=5

Figure 1: RON exhibits MSP-independent tyrosine phosphorylation in 10A cells(a) GFP-positive cells were selected by FACS analysis to yield a 10A/Vector control cell line and RON-expressing cells. RON-expression levels were detected by Western blot with an anti-RON antibody. This antibody recognizes the three different processed forms of RON: the 170 kDa unprocessed form, the 150 kDa processed and phosphorylated form and the 145 kDa processed and unphosphorylated form. Total MAPK level was used as a loading control. (b) 10A/Vector and 10A/RON cells were immunoprecipitated with an anti-RON antibody and subjected to a Western blot analysis for phospho-tyrosine (4G10 antibody). The blot was reprobed for total RON expression. (c)The same experiment as in (b), blotting with antibodies against catalytically activated RON (pY1238/Y1239), and specific tyrosine phosphorylated docking sites in the C-terminus of RON (pY1353, pY1360). (d) Semi-quantitative RT-PCR detected MSP mRNA levels. COS1 cells were transfected with a vector containing an MSP cassette as a positive control. These experiments were repeated at least three times, and shown here are representative images.
Mentions: Endogenous levels of RON were low in the relatively normal epithelial cell line, MCF-10A. Consequently, the cells did not respond to MSP in any biological or biochemical assays we have tested to date. Therefore, to examine the contributions of RON to the progression of breast cancer, we transduced MCF-10A cells with a retrovirus expressing wild-type human RON and IRES-promoted GFP. Pools of infected cells were selected for high, medium and low GFP expression levels by FACS, and, as expected, GFP levels were mimicked by expression levels of RON (Figure 1a). We designated these cells as 10A/RON and our vector-only negative control cell line was designated 10A/Vector. The levels of RON expression in 10A/RON cells were well within the range of expression found in human breast carcinomas, some of which had RON levels that were up to 72-fold higher than the level found in benign tissue (Maggiora et al., 1998). High, medium and low 10A/RON cell lines behaved similarly in all of our experiments therefore, we utilized the medium-RON expressing MCF-10A cell line throughout this study.

Bottom Line: In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors.However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells.Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY, USA.

ABSTRACT
The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

Show MeSH
Related in: MedlinePlus